Single-cell epigenomics reveals mechanisms of human cortical development

During mammalian development, differences in chromatin state coincide with cellular differentiation and reflect changes in the gene regulatory landscap

Transcriptome and regulatory maps of decidua-derived stromal cells inform gene discovery in preterm birth

While a genetic component of preterm birth (PTB) has long been recognized and recently mapped by genome-wide association studies (GWASs), the molecular determinants underlying PTB remain elusive. This stems in part from an incomplete availability of functional genomic annotations in human cell types relevant to pregnancy and PTB. We generated transcriptome (RNA-seq), epigenome (ChlP-seq of H3K27ac, H3K4mel, and H3K4me3 histone modifications), open chromatin (ATAC-seq), and chromatin interaction (promoter capture Hi-C) annotations of cultured primary decidua-derived mesenchymal stromal/stem cells and in vitro differentiated decidual stromal cells and developed a computational framework to integrate these functional annotations with results from a GWAS of gestational duration in 56,384 women. Using these resources, we uncovered additional loci associated with gestational duration and target genes of associated loci. Our strategy illustrates how functional annotations in pregnancy-relevant cell types aid in the experimental follow-up of GWAS for PTB and, likely, other pregnancy-related conditions.Peer reviewe

Characterizing the epigenomic landscape of the developing human cortex at single-cell resolution

During mammalian development, chromatin state differences coincide with cellular differentiation and reflect changes in the gene regulatory landscape. In the developing brain, cell fate specification and topographic identity play important roles in defining cell identity and confer selective vulnerabilities to neurodevelopmental disorders. To identify cell type specific chromatin accessibility patterns in the developing human brain, we used a single cell assay for transposase accessibility by sequencing (scATAC-seq) in primary human forebrain tissue samples. We applied unbiased analyses to identify genomic loci that undergo extensive cell type- and brain region-specific changes in accessibility during neurogenesis and an integrative analysis to predict cell type specific candidate regulatory elements. We found that cerebral organoids recapitulate most putative cell type-specific enhancer accessibility patterns but lack many cell type specific open chromatin regions found in vivo. Systematic comparison of chromatin accessibility across brain regions revealed an unexpected diversity among neural progenitor cells in the cerebral cortex and implicate retinoic acid signaling in the specification of prefrontal cortex neuronal lineage identity. Together, our results reveal the important contribution of chromatin state to the emerging patterns of cell type diversity and cell fate specification and provide a blueprint for evaluating the fidelity and robustness of cerebral organoids as a model for cortical development

Individual human cortical progenitors can produce excitatory and inhibitory neurons

The cerebral cortex is a cellularly complex structure comprising a rich diversity of neuronal and glial cell types. Cortical neurons can be broadly categorized into two classes-excitatory neurons that use the neurotransmitter glutamate, and inhibitory interneurons that use γ-aminobutyric acid (GABA). Previous developmental studies in rodents have led to a prevailing model in which excitatory neurons are born from progenitors located in the cortex, whereas cortical interneurons are born from a separate population of progenitors located outside the developing cortex in the ganglionic eminences1-5. However, the developmental potential of human cortical progenitors has not been thoroughly explored. Here we show that, in addition to excitatory neurons and glia, human cortical progenitors are also capable of producing GABAergic neurons with the transcriptional characteristics and morphologies of cortical interneurons. By developing a cellular barcoding tool called 'single-cell-RNA-sequencing-compatible tracer for identifying clonal relationships' (STICR), we were able to carry out clonal lineage tracing of 1,912 primary human cortical progenitors from six specimens, and to capture both the transcriptional identities and the clonal relationships of their progeny. A subpopulation of cortically born GABAergic neurons was transcriptionally similar to cortical interneurons born from the caudal ganglionic eminence, and these cells were frequently related to excitatory neurons and glia. Our results show that individual human cortical progenitors can generate both excitatory neurons and cortical interneurons, providing a new framework for understanding the origins of neuronal diversity in the human cortex

lentiMPRA and MPRAflow for high-throughput functional characterization of gene regulatory elements.

Massively parallel reporter assays (MPRAs) can simultaneously measure the function of thousands of candidate regulatory sequences (CRSs) in a quantitative manner. In this method, CRSs are cloned upstream of a minimal promoter and reporter gene, alongside a unique barcode, and introduced into cells. If the CRS is a functional regulatory element, it will lead to the transcription of the barcode sequence, which is measured via RNA sequencing and normalized for cellular integration via DNA sequencing of the barcode. This technology has been used to test thousands of sequences and their variants for regulatory activity, to decipher the regulatory code and its evolution, and to develop genetic switches. Lentivirus-based MPRA (lentiMPRA) produces 'in-genome' readouts and enables the use of this technique in hard-to-transfect cells. Here, we provide a detailed protocol for lentiMPRA, along with a user-friendly Nextflow-based computational pipeline-MPRAflow-for quantifying CRS activity from different MPRA designs. The lentiMPRA protocol takes ~2 months, which includes sequencing turnaround time and data processing with MPRAflow

Single-cell epigenomics reveals mechanisms of human cortical development.

During mammalian development, differences in chromatin state coincide with cellular differentiation and reflect changes in the gene regulatory landscape1. In the developing brain, cell fate specification and topographic identity are important for defining cell identity2 and confer selective vulnerabilities to neurodevelopmental disorders3. Here, to identify cell-type-specific chromatin accessibility patterns in the developing human brain, we used a single-cell assay for transposase accessibility by sequencing (scATAC-seq) in primary tissue samples from the human forebrain. We applied unbiased analyses to identify genomic loci that undergo extensive cell-type- and brain-region-specific changes in accessibility during neurogenesis, and an integrative analysis to predict cell-type-specific candidate regulatory elements. We found that cerebral organoids recapitulate most putative cell-type-specific enhancer accessibility patterns but lack many cell-type-specific open chromatin regions that are found in vivo. Systematic comparison of chromatin accessibility across brain regions revealed unexpected diversity among neural progenitor cells in the cerebral cortex and implicated retinoic acid signalling in the specification of neuronal lineage identity in the prefrontal cortex. Together, our results reveal the important contribution of chromatin state to the emerging patterns of cell type diversity and cell fate specification and provide a blueprint for evaluating the fidelity and robustness of cerebral organoids as a model for cortical development

Temporally distinct 3D multi-omic dynamics in the developing human brain

The human hippocampus and prefrontal cortex play critical roles in learning and cognition1,2, yet the dynamic molecular characteristics of their development remain enigmatic. Here we investigated the epigenomic and three-dimensional chromatin conformational reorganization during the development of the hippocampus and prefrontal cortex, using more than 53,000 joint single-nucleus profiles of chromatin conformation and DNA methylation generated by single-nucleus methyl-3C sequencing (snm3C-seq3)3. The remodelling of DNA methylation is temporally separated from chromatin conformation dynamics. Using single-cell profiling and multimodal single-molecule imaging approaches, we have found that short-range chromatin interactions are enriched in neurons, whereas long-range interactions are enriched in glial cells and non-brain tissues. We reconstructed the regulatory programs of cell-type development and differentiation, finding putatively causal common variants for schizophrenia strongly overlapping with chromatin loop-connected, cell-type-specific regulatory regions. Our data provide multimodal resources for studying gene regulatory dynamics in brain development and demonstrate that single-cell three-dimensional multi-omics is a powerful approach for dissecting neuropsychiatric risk loci